Prevention and remediation of petroleum reservoir souring and corrosion by treatment with virulent bacteriophage

ABSTRACT

Petroleum reservoir souring caused by microbially induced production of hydrogen sulphide and other sulphur compounds and the attendant corrosion are remediated by isolating bacteriophage specific for the problematic bacteria (target bacteria) and adding an effective amount of such bacteriophage to water introduced into or resident in the reservoir to kill at least some of the target bacteria. Suitable virulent bacteriophage may be indigenous in the water or located in surrounding areas or taken from a known banked stock. Means of concentrating solutions of bacteriophage are also disclosed.

RELATIONSHIP TO OTHER APPLICATIONS

This application is a Divisional application of U.S. application Ser.No. 12/983,136 filed Dec. 31, 2010 and claims benefit of ApplicationSer. No. 61/295,142 filed Jan. 14, 2010.

FIELD OF THE INVENTION

This invention relates to control of souring (hydrogen sulfideproduction) and corrosion in oil and gas reservoirs caused by bacteria.More specifically, it relates to control of bacteria that produce acidand/or sulfur compounds that cause reservoir souring, fouling andcorrosion; control being effected by destruction of targeted bacteriawith naturally occurring bacteriophage, virulent for targeted bacteria,particularly sulfate reducing bacteria (SRB) and acid producing bacteria(APB).

BACKGROUND

Microorganisms, including bacteria, are ubiquitous in nature and canhave profound negative effects on oil and natural gas recovery.Bacterial fouling of the water needed to hydrofracture (“frac”)reservoir rock or to “water-flood,” to increase production of oil andgas, can contaminate or “sour” the reservoir by producing hydrogensulfide (H₂S). This decreases the value of the product and can makemarginal wells unprofitable. Sulfate reducing bacteria (SRB) producetoxic, flammable H₂S, which shortens the lifetime and lowers thereliability of any piping and tankage, and introduces additional safetyrisks from drill rig to refinery. Acid producing bacteria (APB) produceacids, including sulfuric acid, which lead to additional corrosion.

Bacterial fouling leads to serious problems in the oil and gas industry.Bacterially-evolved hydrogen sulfide sours petroleum reservoirs,elevating risk and devaluing the product, while bacterial production ofiron sulfide creates black powder accumulation, causing pipelineblockages. Microbially-influenced corrosion attacks the whole system,from fracture tank to refinery, and degrades fracture fluid additives.In Barnett Shale operations in Texas, water is typically stored in largeponds which are open to the atmosphere prior to the start of fracturingwork, allowing the water to become heavily contaminated with bacteria.In addition, bacteria become established in biofilms near the wellboreduring shut-in of the well.

The Barnett Shale formation's low permeability requires the use oflarge-volume hydraulic fracturing technologies to enhance gasproduction. Other shale formations, such as the Marcellus in the easternU.S., also require hydraulic fracturing. In a typical “frac” operation,water is collected in portable tanks or large, purpose-dug ponds from avariety of sources, including water wells pumping from aquifers,chlorinated city water supplies, and ponds, rivers, and lakes. Each ofthese water sources has some level of innate indigenous bacterialcontamination that continues growing during the collection reservoirs'exposure to the atmosphere.

Hydrofracturing (“fracing”) and “water flooding” is heavily dependent onthe availability of water, and a typical horizontal “frac” operationrequires one to five million gallons of water. The water is pumped intoa production well at very high rates (one to over two hundred gallonsper minute (gpm). Droughts such as that affecting the Barnett shaleoperational area have been common over the past several years. Duringtimes of drought, water recovered from previous hydro-fractureoperations (“flow-back” or “produced” water) is reused, and mixed with“fresh” water in holding ponds or tanks. This reused water introduceselevated bacterial fouling concentrations and solids loadings. Even intimes when no drought exists, the universal use of flow-back water inall “frac” operations is utilized to mitigate the expense andenvironmental harm done in removing and disposing the highlycontaminated waste water and is increasingly being required byregulation.

To counter bacterial fouling and reservoir souring, chemical biocides,commonly hypochlorite bleach, are applied to the fracture water. Thecost of the biocide treatment for a single typical “frac” operation canbe as much as $50,000. Additionally, the design of recovery systems withsour service alloys, thicker pipe, and heavier valves leads to increasesin capital expense.

The scale of the problem is enormous. The Barnett Shale undergroundnatural gas formation extends over 5,000 square miles in north centralTexas. A total of 6,519 gas wells with a further 4,051 permittedlocations existed as of Aug. 15, 2007. Wells are being drilled withinpopulated areas, such as the Dallas-Fort Worth city limits, where it isvital to minimize risk and environmental impact. The petroleum industrycurrently spends $2 billion on biocides annually. Broad spectrumbiocides require the additional expenditures associated with regulatorycompliance. These biocides may remain in the water when it is pumped outof the well, creating waste handling and disposal problems.Understandably, biocide usage in the petroleum industry is facinggrowing regulatory resistance because of the negative impact on theenvironment and associated health risks.

As well as requiring enormous expenditures, biocides are notsufficiently effective. Any bacteria that are endemic or are introducedinto the formation encounter favorable growth temperatures andconditions during the “frac” and flooding operations, as the largevolumes of water pumped downhole result in near wellbore cooling. Wellsmay be shut in following the operation while surface processingequipment and flowlines are installed, leaving time for bacteria tocolonize. Once bacteria become established in a well, they developbiofilms that supply a stream of bacterial contamination downstream thewell through water tanks, flow lines and disposal facilities. Biofilmsprotect the bacteria from the chemical biocides and a program ofregular, high volume biocide application must be initiated merely tokeep the free-swimming bacteria in check and minimize problem bacterialbyproducts. Biofilms themselves are impervious to biocides, and can onlybe mechanically scoured, as with pipeline “pigs”. In addition, there isincreasing biocide resistance being observed in hydro-fracture and floodwater bacteria.

Other reservoirs are “flooded” with water to enhance recovery—usuallyoil recovery. In “water flood” operations, injection wells are drilledinto the producing horizon and water is pumped—as in fracturing—todisplace the oil and/or gas through a formation into other “recovery”well(s) in the same field. Since the water is injected into thereservoir is contaminated with bacteria, similarly to the water used for“fracing,” the same problems of souring, fouling, and corrosion occur.

Bacteria also cause a host of additional problems in other sectors ofthe petroleum industry. Another potential “expense” is the social costof catastrophic failure. Microbiologically-induced corrosion (MIC) hasbeen a factor in several major oil and gas pipeline incidents, includingthe well-publicized 2006 Alaska Pipeline spill. MIC occurs on theinsides of pipes or storage vessels, and especially under biofilms.

A better control strategy would be: inexpensively manufactured,environmentally benign, adaptable to changing microorganisms to preventresistance, targeted towards those microorganisms that constitute thethreat, and capable of penetrating and destroying biofilms. Such acontrol strategy would optionally be able to sense and adjust to thedifferent concentrations of microorganisms encountered, even within thewell. The present invention is just such a strategy, providing bacterialcontrol based on bacteriophages, the natural predators of bacteria.

SUMMARY OF THE INVENTION

The present invention is a safe, natural, environmentally sound means ofcontrolling bacterial contamination, corrosion, fouling and souring ofoil and gas wells and reservoirs that result from injectingbacteria-contaminated water into a well. More specifically, in oneembodiment, the invention is a process for remediation of biofouling andsouring of petroleum reservoirs and coalbeds comprising: adding to thewater used in flooding and “fracing” operations an effective amount ofvirulent bacteriophages specific for APB and/or SRB found in the waterused in the well. These phages may be produced by concentrating anaqueous solution of virulent bacteriophages from bacteria indigenous tothe water. In another embodiment, the invention is a process forreplication of bacteriophages comprising: a vessel with an inlet for anaqueous solution containing target bacteria; an inlet for an aqueoussolution of bacteriophages virulent for target bacteria; an outlet for asolution containing replicated bacteriophage; wherein the flow rate ofthe inlet solution containing target bacteria and the flow rate for thesolution containing bacteriophages virulent for target bacteria areadjusted to obtain substantially complete destruction of the targetbacteria. Other more specific embodiments are disclosed in the DetailedDescription of the Invention. The phage-based bacterial controltechnology of this invention will improve operational efficiencies andprolong the operational life of marginal wells that would ordinarilyhave been withdrawn from service. It will also decrease the capitalcosts of creating new wells by maintaining sweet gas production,mitigating the need for sour service pipes and hydrogen sulfide removalapparatus. The ability to universally recycle flowback water willdecrease the cost and environmental impact of “frac” and floodingoperations. An ancillary benefit will be the improvement of results from“frac” and flooding operations.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the process of the invention.

FIG. 2 is a diagrammatic representation of one aspect of the process ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

Current customary methods of microbial control in oil and gas formationsand wells apply broad spectrum biocides, typically by injection into thestream of fracture or flood water at a blender prior to injection intothe well. Customary biocides include glutaraldehyde,glutaraldehyde/quaternary ammonium compound blends, isothiazolin,tetrakis(hydromethyl) phosphonium sulfate (THPS),2,2-dibromo-3-nitrilopropionamide, and bronopol. However, these biocidesoften have major health risks to humans and all animals in the foodchain. THPS and hypochlorite bleach are the most commonly usedantimicrobials in the Barnett shale operations area. These EPAregistered biocides cannot be introduced into an open pond, as they willpermeate into the groundwater, killing aquatic organisms and frequentlybeing consumed by terrestrial animals. All biocides in use by theindustry are intentionally employed in a broad spectrum manner. Theireffectiveness is determined by conventional culturing methods whichmerely determine the presence of bacteria and make no attempt todetermine genus or species. These methods are labor and materialintensive and are essentially unchanged in the past forty years. Intypical biocide assessment practices, samples of “frac” water arediluted and cultured in specialized growth medium under variousconditions, with and without biocide, for various lengths of time andthen compared for bacterial cell density, resulting in more than 40 testcultures each time. There are seasonal variations in bacteria, requiringdifferent growth and test conditions, to which the bacteria may responddifferentially. The results take days and, thus, cannot be used forrapid optimization of biocide application. “Water scarcity leads to thereuse of hydrofracture water but when hydrofracture ponds are filledwith this already bacterially-contaminated water at the last minute, theassays cannot return results on bacterial activity prior to the waterbeing pumped into the well. The typical field solution to thisuncertainty is to apply massively excessive concentrations of sodiumhypochlorite. A useful alternative to this would be the instantaneous,on-line determination of bacterial concentrations directing the biocideadministration.” (Use of Microbiocides in Barnett Shale Gas WellFracturing Fluids to Control Bacteria Related Problems; J. K. Fisher, K.Johnson, K. French and R. Oden, Paper 08658, NACE, International; 2008Corrosion Conference and Expo.)

The present invention is a process of controlling the problem bacteriaby using virulent bacteriophage, instead of synthetic biocides.Bacteriophages are the ubiquitous and natural viruses which infect, arereproduced within, and lyse bacteria. Phage infection is initiated whenthe tail proteins recognize and adsorb to specific cell surface featuresof the target bacterial host. This triggers the injection of the phageDNA into the bacterial cytoplasm. The genes in that DNA are expressed bythe bacterium's own protein synthesis apparatus, resulting in thesynthesis and assembly of approximately 30 to 100 progeny phageparticles over the course of minutes to several hours. After, typically,15 to 60 minutes, the cell explodes (“lysis”) as a result ofphage-encoded lytic enzymes, liberating hundreds of progeny phage thatcan then adsorb to new bacterial hosts and repeat the process. In thismanner, bacteriophages replicate themselves. Random environmentalsamples indicate the presence of 10-100 phages for every bacterial cell,indicating 10³⁰-10³¹ phages in the biosphere.

There are important consequences to this life cycle. First, given agrowing bacterial culture to attack, phages can proliferate atunimaginable rates. Within two hours of the addition of a singleparticle of the classical phage T7 to a laboratory culture of 10 billionEscherichia coli cells, more than 99.9% of the bacteria are destroyedand 10 trillion virus particles are generated. There is, thus, ascientific basis for calling bacteriophages “the only medicine thatgrows” and, in fact, many of the early myths of curative springs orrivers were grounded in the reality that phages existed in these watersat concentrations capable of curing leprosy or cholera (Hankin, 1896).Second, phages are specific for target (or matching) bacteria, becausethey generally only bind to the type of bacterium that their adsorptiondevice, or “tail”, recognizes, and that is encoded in their DNA. Thus,phages are harmless to other bacteria and, obviously, to higherorganisms.

Phages do not infect plants or animals and are, therefore, safe toproduce, store, handle and apply. Bacteriophages have been declared“Generally Recognized as Safe” for use in human food.

Because bacteriophages reproduce along with the microorganisms that theyinfect, in the method of this invention, once down-well, they willspread to other bacteria of the same species.

The viscosity additives to “frac” and “flood” water are, ironically,food sources for fouling bacteria. Bacterial degradation of theviscosity additives occurs early in the fracture water tanks and in thesubsurface, causing premature viscosity drops and fracture closing. Whena panel of phage cocktails is mixed into the fracture fluids, thesepremature failures may be abated or avoided, forestalling otherwiserapid decline of the wells and retaining production rates. Phageproducts may also impart a level of immunity to the reservoir, therebyextending the commercial life of a producing well.

In one embodiment of this invention, as more fully detailed below, highconcentration virulent phage solution is first injected into a well,followed by the bulk of the “fracing” water, thus enhancing the immunityof the reservoir by filling the fractures with the highest level ofvirulent phage biocide. Where the “fracing” is conducted in stages orsegments—where a segment of the well is fractured (after perforating ifthere is casing) and then temporarily sealed—higher concentrations ofphages will be injected into each segment as the segment is fractured.High concentration of phages is highly desirable to initiate infectionof problematic bacteria.

Other petroleum resources will be similarly positively impacted.Petroleum reservoirs will be prevented from souring during water floodoperations, as phage products will kill SRB and impart a persistentlevel of immunity to the reservoir. Phages can also be used to treatexisting reservoirs. Thus, the use of phages in accordance with thisinvention will reduce completion costs, workovers and re-completions,and, most importantly, overall production costs due to capital andoperational expenses associated with bacterial fouling, corrosioncontrol and repairs.

As used herein, the following definitions apply: A phage cocktailincludes multiple, receptor independent phages for each target bacterialhost. This is different from a phage panel, which is a collection ofphages chosen to cover as wide a host range as possible. For thepurposes of this invention, the phage treatment of waters will consistof a panel of phage cocktails, that is, there will generally be at leasttwo phage cocktails for each of several target SRB bacteria, and twovirulent phages for each target bacteria. Since some SRB phages areknown to be polyvalent—effective against more than one strain ofSRB—there may not need be a separate cocktail for every strain of targetbacteria. This panel of cocktails is designated herein as phage“multi-panel”.

A somewhat typical flow scheme for a “fracing” operation (as, forexample, in a Barnett Shale gas well) may be understood by reference toFIG. 1. Water from a lined storage pit 200 is pumped into one of several500 bbl temporary storage tanks, 201, 202 and 203, or the tanks arefilled directly from other water sources. Water in the storage pit maybe tanked in, produced from water well(s), river water, natural run offwater, or any other convenient source. For reference, a half acre pit of6 ft average depth contains 488,779 gallons. Most of the water sourceswill be heavily contaminated with bacteria. Since the pit is open itwill have additional air-borne and run-off bacterial contamination withnumerous and varied bacterial strains.

Water from the temporary storage tanks is mixed with chemical additivesand proppants, to hold the fractures open (usually sand or ceramicbeads), and with biocides from tank 204 (usually a tank truck). Waterand additives are mixed in mixer 220 and picked up by high pressurepump(s) 212 for high pressure injection into a wellbore 300. This highpressure water causes fractures or cracks in the gas bearing rock orshale formation allowing the gas to be released to exit the well throughthe well bore. The proppants help hold the fractures open.

The well bore 300 is sealed up-well of the to-be-fractured area bypacker(s), 301, to maintain pressure in the wellbore during “fracing”.Water pumping rates range from about 10 bbl/minute to as much as 200barrels/minute (420-8,400 gal. per min.). Rates of 70-80 barrels/minuteare typical in Barnett Shale wells. The “frac” water may be injected inone or more stages, or may be injected into individual segments of thewell bore. For example, the segment of deepest portion of the well maybe sealed and fractured, then filled with sand and the tools pulled backto seal and fracture a second segment, and so on. For the purposes ofthis invention, each of these segments may be considered a separate“frac” operation.

After the desired amount of water has been pumped into the well forfracturing, the well sits idle while production equipment is installedat the well head. Thus, the well may sit with “frac” water in it fordays or months. During this time bacteria grow and produce acid andsulfur compounds in the reservoir formation. Moreover, these bacteriallyproduced compounds will cause further problems when the water isreturned to the surface, including microbially induced corrosion (MIC)of top side equipment. When the surface production equipment isinstalled, the injected water is allowed to return (flowback and“produced” water) to the surface for disposal, shown in FIG. 1 as storedin tank 206. In “fracing” operations, generally about 20-40% of theinjected water remains in the formation. The flowback or “produced”water contains oil, salts, contaminates, and increased bacterialconcentrations, and is generally problematic for recycle. In a study ofbiocides in several Barnett Shale wells, the bacteria level increased atleast one order of magnitude from the source water, e.g. from 1×10⁶bacteria/ml to 1×10⁷ pfu/ml. Increasingly, recycle, and treatment of the“produced” water, is required.

According to this invention, bacterial control is accomplished by addingan effective amount of virulent bacteriophage solution, as in a phagemulti-panel, to the “frac” water as, for example, by adding it to thewater storage pit (pond) or temporary storage tanks. In water floodoperations, to enhance production, or in an already soured reservoir,the bacteriophages will be added to the water or injected with the waterto combat SRB, and will prevent further degradation and production ofH₂S. If phage multi-panel is added to the “frac” water pond, it ispreferred that it be added under the surface of the water (SRB areanaerobic), and added slowly in discrete locations—such as can beaccomplished with a soaking hose or similar injection means. If thephage multi-panel is distributed in discrete volumes, rather than byrapid mixing, the phage concentration level remains high in the areaimmediately surrounding the injection for sufficient time to allowphages to infect target bacteria. The rate at which phages attach to,infect, and lyse target bacteria is highly dependent upon theconcentration of phages and target bacteria. Generally, it is desiredthat each be at least about 1×10⁵ pfu/ml. By slowly seeping the phagemulti-panel into the water, the phages form a pocket of highconcentration surrounded by concentrated bacteria. As the phages infectand lyse target bacteria, the number of phages multiply exponentially;thus, the phages diffuse through the water in a kind of wave ofconcentrated phage attack of the surrounding bacteria. If, on the otherhand, the phage multi-panel is rapidly and thoroughly mixed with the“frac” water in large volumes, the concentration is greatly reduced andthe phage infection of bacteria is slowed to unacceptable levels. Thus,a means of slowly adding the phage solution, such as a type of “soakinghose”, is desirable.

Because the quantity of water is so great, large amounts ofbacteriophage solution will be needed if the entire “frac” water is tobe treated. Moreover, phages are not mobile—they have no mechanism formoving about to find and attack target bacteria. Phages must be broughtinto physical contact with target bacteria. Therefore, in oneembodiment, the needed virulent phages are generated on-site. This isaccomplished in a phage proliferator/concentrator process.

Phage Proliferation and Concentration

In order to produce a sufficient amount of bacteriophages to treat thelarge volume of water, phages may be replicated and concentrated onsite. Fortunately, propagation of virulent phages is achieved by thebacteriophage injecting itself into its matching bacteria andreplicating itself at the same time as it destroys the bacteria—as isalso explained in more detail below. Bacteriophage proliferation for usein treating the “frac” or “flood” water is illustrated in FIG. 1. Ingeneral it is preferred, and sometimes necessary for the entireproliferation/concentration system to be blanketed with a non oxygengas. Nitrogen is preferred, since the SRB are aerobic and will be killedif there is significant oxygen in the system.

Vessel 101 is a proliferator/concentrator. Water containing targetbacteria is pumped into vessel 101 through valve 120, where it is mixedwith bacteriophage panel or multi-panel virulent for the targetbacterial strains, shown as being pumped out of vessel 104 with pump 110through valve 124 to be mixed with the incoming bacteria-containingwater. Some forms of SRB will be substantially destroyed by theirspecific virulent phages in less than 20 minutes. The concentratorvessel is sized to provide a flow rate of concentrated bacteriophagesolution sufficient to treat the desired volume of “frac” water. A 4 ftdiameter vessel will have a volume of 12.6 ft3/ft of height. A 6 ftdiameter vessel will have 28.3 ft3/ft. Thus, a 4 ft diameter vessel, 8ft tall, will contain 100.8 ft3′ and a 6 ft diameter vessel, 8 ft tall,will contain 226.4 ft3.

A flow rate of 9.3 gpm in the 4 ft. diameter reactor, and 37.7 gpm inthe 6 ft. diameter reactor, will provide 20 minute residence time(equivalent to the time needed for a substantially complete kill of somestrains of SRB bacteria). Residence time will, of course, vary with flowrate, and can be suitably adjusted to provide sufficient time for phagereplication for each phage species.

Concentration of bacteriophage in solution leaving vessel 101 dependsupon the concentration of target bacteria in the incoming water. Whenmatching bacteria are present, some phages may be replicated by a factorof about 20:1. Therefore, for example, when the incoming water contains2×10⁶ pfu/ml, the outgoing stream will contain 4×10⁷ pfu/ml. If thereplication is 100:1, then the outlet stream will have a phageconcentration of 1×10⁸ pfu/ml—a two orders of magnitude increase. Thephages will continue replicating themselves so long as a sufficientconcentration of target bacteria remains in the water. Thus, thereplication will continue when the outgoing concentrated phage solutionis mixed with bacteria-containing “frac” water.

Initially, the proliferator/concentrator is fed with a solution ofbacteriophage multi-panel (mixture of virulent phages) that has beenseparately generated—shown in vessel 104 and passed to theproliferator/concentrator through valve 124 by pump 110. Once theconcentrator is functioning, phages may be supplied by recycle of aportion of the output stream through valve 128. The amount of recyclewill preferably be sufficient to provide a phage-to-target-bacteriaratio between 1 to 0.001. In general, 10 the recycle will contain about20 times the concentration of phages as the concentration of targetbacteria in the source water since some SRB phages will replicatethemselves in target bacteria about 20:1. Some of the concentrated phagesolution may be stored for future use, as in one of the temporarystorage tanks, 201, 202, and 203.

In FIG. 1, vessels 102 and 103 are used for culturing target bacteriawhich may optionally be added to the proliferator/concentrator 101 toincrease the concentration of incoming bacteria and, hence, the amountof phages produced. Such supplemental bacteria may also be varied togenerate a desired concentration of phages in the output stream.Culturing of bacteria may be conducted on-site or at an off-site,centralized location and brought to the treatment site. Alternatively,target bacteria may be concentrated from the “frac” water or othersource in a tangential flow filter system. Such a system is illustratedin FIG. 2.

Referring to FIG. 2, water is pumped from the “frac” water pond 200 (orother suitable storage, as would be required in some water floodoperations) by pump 305 to filter 304—a coarse filter to remove largerparticles and trash. From filter 304 the water passes by conduit 321 totangential flow filter 301, having a filter screen, 302, of about 0.2micron. The screen is sized to hold back SRB and let smaller particlespass. The filter water may be recycled to the filter by pump 310(through conduit 322). The filtrate passes to tank 306, where it may bedirected as needed by conduit 320.

The illustration in FIG. 2 shows the water source as the “frac” waterpond. It may, of course, be any suitable source. In one embodiment, thesource will be the “produced” water from the well (see vessel 206 inFIG. 1). In general, the “produced” water will contain salt (NaCl) andsome target bacteria, which may be halophilic. If it is found that theoffending target bacteria are halophilic, and target SRB from the “frac”water does not survive in the salt water environment of the formation,it will be desirable to isolate target halophilic bacteria recoveredfrom the reservoir. Such bacteria can also be cultured, as describedabove, by using a brine culture solution.

In FIG. 1, vessel 101 contains phages virulent against the target orhost bacteria used to start the process. It may be replenished from theconcentrated outflow of the phage concentrator 101 or from an externalsource.

Thus, in operation, the phage concentrator will take in “frac” waterfrom one of the storage tanks 201, 202 or 203 through valve 120 or,alternatively, directly from storage pit 200 through valve 125.Concentrated phage solution may pumped to one of the temporary workingtanks 201-203 through valve 121, or returned to the water storage pit200 through valve 126. In either case, the phages will continuereplicating if there are sufficient target bacteria present,substantially destroying most of the target bacteria.

In one embodiment, more concentrated phage solution will be first pumpedinto the well before the bulk of the “frac” water is injected. Forexample, if the bulk of the treated “frac” water contains 1×10⁶ virulentpfu/ml, the first solution will be about 1×10⁷ to 1×10¹⁰ pfu/ml. Thisallows the fracture formation to be saturated with a “packet” of highconcentration virulent phage solution to mitigate bacterial growth inthe well. In general, this first solution will be about 0.01 to 10%,preferably about 0.1 to 5%, of the total “frac” water injected.

All the vessels 101, 102, 103, and 104 are constructed of simplematerials. They only need to be sufficiently strong to hold thesolutions. Corrosion is not a particular problem, although they shouldbe able to contain oily “flowback” water, which will include salt andchemical additives. It is desirable that they be able to be washed andsterilized with bleach solution. It is also desirable that they be ableto be “blanketed” with a non-oxygen gas. Generally, most plasticmaterials used for tanks and vessels are suitable, including fiberglass,polypropylene, polyvinyl chloride, and polyurethane. Stainless steelwill also be suitable. Other commercially available materials will beobvious to those skilled in the art.

Since bacterial growth, and to some extent phage proliferation, istemperature sensitive, there is provided in one embodiment means forheating either the inlet streams to the vessels or heating the contentsof the vessels. The streams may be heated by heat exchange, electricalheaters, or any other suitable means known in the art. The contents ofthe vessels may be heated with electrical or steam heaters or othersuitable heating means known in the art.

These vessels are not especially heavy, and the equipment is notextensive; therefore, in one embodiment the proliferation/concentratorequipment—vessels 101, 102, 103, and 104, and associated pumps, valvesand piping—are mounted on a movable platform so that they can easily betransported from well site to well site. These can be mounted on skids(that can be lifted onto a truck bed), or on a trailer or truck bed.

In another embodiment, this invention is a composition comprising aconcentrated amount of bacteriophage virulent for prokaryotes, i.e. APBand/or SRB found in water sources used in oil, gas, and coalbed wellflooding and “fracing”. The composition will contain between 1×10⁴ to1×10¹² pfu/ml. In one embodiment, the target bacteria are SRB.

Identifying target bacteria and virulent phages and culturing bacteriaand large scale bacteriophage are necessary steps in this invention andexplained more fully below.

1. Identifying Target Bacteria:

Target bacteria are identified by sampling the source water and/orbiofilm. From samples, the target bacteria can be isolated andcharacterized, to some extent based on what is generally already knownabout the causes of corrosion, souring and fouling. From these samples,virulent bacteriophages are identified for target bacteria andbio-corrosive organisms, i.e. SRB and APB. Sufficient phages are thenisolated to effectively lyse the target bacteria, and an effectiveamount of phage solution is added to the water used for “fracing” a wellformation. SRB comprise Desulfovibrionaceae selected from the groupconsisting of D. vulgaris, D. desulfuricans and D. postgatei. In yetanother aspect, the bio-corrosive organisms comprise Caulobacteriaceaeselected from the group consisting of C. Gallionella and Siderophacus. Awide variety of organisms may be targeted, e.g., archaebacteria,eubacteria, fungi, slime molds, and small, bio-corrosive organisms. TheSRB group of bacteria reduces sulfates to sulfides, releasing sulfuricacid and hydrogen sulfide as byproducts which react with iron to formthe characteristic black precipitate iron sulfide. Hydrogen sulfide gasis not only extremely toxic and flammable, but it causes souring of thepetroleum product, resulting in reduced quality and increased handlingcost. The term “SRB” is a phenotypic classification, and severaldistinct lineages of bacteria are included under this umbrella term.Target bacteria include members of the SRB including, withoutlimitation, members of the delta subgroup of the Proteobacteria,including Desulfobacterales, Desulfovibrionales, andSyntrophobacterales. Also targeted are the APB bacteria that produceacidic metabolites. This specifically includes sulfur-oxidizing bacteriacapable of generating sulfuric acid. This includes, without limitation,sulfur bacteria such as Thiobacilli, including T. thiooxidans and T.denitrificans. Targeted bacteria further may include bacteriapopulations and isolates, and further includes corrosion-associatediron-oxidizing bacteria. Also included are isolates of theCaulobacteriaceae including members of the genus Gallionella andSiderophacus.

Still further, bacterial populations may work synergistically with thebio-corrosive bacteria described above. These include members ofmicrobial consortia exhibiting biofilm formation activity. Such biofilmscan provide the anaerobic microenvironment required for the growth ofcorrosion promoting bacteria. As such, the target of phage treatment caninclude not just the corrosive metabolite producing bacteria, but alsoany bacteria involved in forming the microenvironment required forcorrosion. Additionally, biofilm producing bacteria involved in thebiofouling process are included in the category of targets for phageremediation. Biofilm forming genera of bacteria include Pseudomonas orVibrio species isolated in affected containment systems.

Bacterial populations responsible for biofilm blockage may also beselected for phage treatment. All bacteria that are to be targeted forphage treatment are part of the selected bacterial subpopulation.

2. Culturing Target Bacterial Strains:

The target bacteria are cultured by means well known in microbiology.Any means of culturing bacteria that promotes growth of the bacterialpopulation are suitable. For example, liquid cultures of D. vulgaris canbe grown in ATCC medium1249 Modified Baar's medium for sulfate reducers.Plate cultures of D. vulgaris are then grown on ATCC medium: 42Desulfovibrio medium. Cultures have been grown at either 22° C. or 30°C. in anaerobic GasPak jars (VWR). D. vulgaris growth forms acharacteristic black precipitate in media containing ferrous ammoniumsulfate, an indicator of sulfate reduction.

Sufficient bacteria can be grown and enriched in a relatively smallcontainer. Therefore, it is preferred that the initial culturing ofbacteria be conducted on site or in-situ, as for example, as illustratedin FIGS. 1 and 2. Larger quantities, as are needed for large scaleproduction of phages, are preferably grown in a centralized locationhaving the equipment and resources needed.

If the target SRB bacteria are halophilic, it will be necessary toadjust the culture by addition of NaCl.

3. Identifying Virulent Phages for Target Bacteria:

The geographic distribution of industrial bacterial contaminations isworld-wide and transverses many geographic and geological boundaries.Similarly, the sources of phages for controlling bacterial infestationsinclude any site where bacteria are found and, thus, transverses manygeographic and geological boundaries. While existing phage stocks willbe screened for activity on target bacteria, new phages will also beisolated from the same site or location where the bacteria pose aproblem, such as soils, stagnant waters, indigenous water and the like.As the natural predators of bacteria, populations of bacterial phageswill be most abundant near abundant sources of their prey. Therefore,the process of identifying phages specific for any bacterial populationis to first identify an environmental site where that bacterial type isabundant. This means that there is not one environment that will serveas a source of phages for all target microbes. Instead, the exactenvironmental sample will vary from host strain to host strain. However,there are general guidelines for identifying the environmental samplemost likely to yield desired phages. An ideal sample is marine orfreshwater sediment from an environment favorable for the growth of thehost bacteria. Specific physiochemical properties of the sediments areimportant. While the exact parameters will vary from host to host,variables to consider include salinity, temperature, pH, nitrogen oreutrophication, oxygen, and specific organic compounds. An example,which is not intended to be a guideline for all protocols, would be theidentification of phages active against a sulfate reducing bacterium(SRB) such as Desulfovibrio. Sediments enriched in SRB are characterizedby a black anoxic layer and the production of odiferous volatiles suchas hydrogen sulfide. These sediments are common in areas experiencingeutrophication in concert with the resulting oxygen depletion.Therefore, a sample likely to possess SRB specific phages will be ablack, hydrogen sulfide producing sediment collected from waters rich inorganic compounds.

The choice of a sample site for phage isolation is customized to aspecific host bacterium. Phage isolation sites may include any body ofwater (natural or man-made), sediments, or soil samples. Phage isolationsites may also include man-made structures such as the target watersource, containment or settling tanks, creeks, and ditches. Within theman-made structures, the sludge-like deposits composed of organic andinorganic sediments that have settled at the bottom of the structuresare often the optimal sampling site for isolation. Phages for any givenhost can be found at the same conditions relative to salinities,temperatures, pH, pressure, nitrogen concentrations, and oxygen levelsthat are favorable to the growth of the host bacteria. Bacteria varygreatly with regard to carbon source utilization, similarly phages thatinfect these bacteria can be found in these environments regardless ofcarbon source being utilized by the bacteria. Similarly, bacteria andphages vary greatly with regard to tolerance and utilization ofindustrial waste materials such as metals, heavy metals, radioactivity,and toxic chemical wastes including pesticides, antibiotics, andchlorinated hydrocarbons.

As an alternative to identifying samples based on physiochemicalproperties, molecular tools are used to identify sediments possessingwild populations of bacteria similar to the target bacteria. Thesemethods typically require some level of purification of DNA from theenvironmental sample, followed by the detection of marker DNA sequences.

The most straightforward of these are polymerase chain reaction (PCR)based technologies that target 16 s rDNA sequences. These can beanalyzed by methods such as denaturing gradient gel electrophoreses(DGGE) or by DNA sequencing.

4. Isolation of Novel Phages Active Against Target Bacteria:

It is necessary to match collected phages to a target strain ofbacteria; matching in the sense of obtaining a phage sample that isspecifically virulent (lethal) for the target bacteria strain. Matchingis accomplished by identifying the bacteria strain and empiricallyapplying a phage sample until a clearing of the bacteria is obtained.Not all bacteria will be destroyed because a minimum level is requiredto initiate infection and clearing. It may also be accomplished withoutever identifying the bacteria strain by empirically finding a matchingvirulent phage from collected or stored phage samples. These empiricalmethods are more research intensive than specifically identifying thebacteria and/or the virulent phages, but are equally effective for thepurpose of this invention.

Using criteria discussed above with respect to the individualcharacteristics of the target bacteria, an appropriate environmentalsite will be identified from which phages can be isolated. The primarymethodology used to isolate these phages is an enrichment method.Sediment, sludge, or soil samples from the environmental site will bemixed with a solution containing salts and peptides. The exactcomposition of this solution can vary but, in general, will approach thesame composition as Lysogeny Broth (commonly referred to as LB media:per Liter—10 g tryptone, 5 g yeast extract, 10 g NaCl).

The ratio of sample to LB will vary, with the goal of producing a thick,turbid sludge. This is shaken for several hours, and a sterile rinsateis produced from it by sequential centrifugations and filtrations toremove solid material greater than 0.2 microns. This is termed a“rinsate” and the rinsate is then supplemented with concentrated freshbacterial media (which will vary depending on the exact bacterial hostbeing grown). A small amount of the host is then added to therinsate/media mix and allowed to incubate for one to several daysdepending on the growth rate of the host. Incubation conditionsincluding shaking, media temperature, and oxygen levels will be thosethat promote growth of that particular host. After incubation,chloroform will be added to 0.01% and the solution will be sterilized bysequential centrifugation and filtration to remove intact bacterialcells. This solution is termed an “enrichment”. Phages in the“enrichment” are assayed for by several different methods including theplaque assay, liquid culture lysis, or visualization by electronmicroscopy.

The final product is an aqueous solution containing phage particles in aweak phosphate buffer with minimal bacterial cellular debris.

5. In-Situ Test of Identified Killer Phage Strains:

Matching of the identified phages and target bacteria or biofilm inisolation is critical to the success of the process of this inventionand must be validated in “real life” conditions of the environment inwhich it is to be used. Thus, the matched phages are tested in the waterconditions that exist. This is suitably done in a side-stream or aliquotof the water system to be treated. A suitable means for this test, forexample in the “frac” water pit, is to pump a stream of the water sourceinto a suitably sized container or side loop for sufficient time toallow it to come to equilibrium with the water source. The identifiedphages are introduced into the stream (either batch wise or incontinuous flow) and tests are made to determine if the population oftarget bacteria is reduced.

6. Preparing Suitable Quantity of Identified Phage Multi-Panel to Treatthe Target Water System:

The treatment phage multi-panel consists of a mixture of virulent phagesthat have been found to “match” target bacteria and biofilm to betreated. Sufficient phage solution must be manufactured to provide aneffective amount and concentration to significantly reduce the targetbacteria population, or at least to initiate phage proliferation in asystem, as described in reference to FIG. 1.

For this, phages exhibiting bacteriolytic activity against targetbacteria will be selected. Phage multi-panels may include pre-existingphage isolates as well as the de novo isolation of novel phages fromsamples taken at the water site. Thus, in one embodiment, the step ofproducing the infective (virulent) phage panel may further includescreening and isolating naturally occurring phage active against theselected bacterial population. In another embodiment, it may beunnecessary to screen for phages where the suspect bacterial populationsare already known or suspected. Phages may be isolated by a number ofmethods, including enrichment methods or any technique involving theconcentration of phage from environmental or industrial samples followedby screening the concentrate for activity against specific host targets.

Additionally, new methods for isolating phages are likely to bedeveloped, and any phages isolated by these methods are also deemedcovered by the claims of this invention. Given the high geneticdiversity of phages, these naturally occurring phages will include thosewith novel genomic sequence as well as those with some percent ofsimilarity to phages known to infect other bacterial clades. Most of thenew phages are expected to be members of the taxonomic groupCaudovirales, also generally referred to as the tailed phage. The use ofphage in an infective cocktail is dependent on the phage's bacteriolyticactivity. Bacteria targeted by treatment with phage or phage panelsinclude any isolates present in the target water system.

Phages can be optimized for effectiveness by selection for naturallyoccurring variants, by mutagenesis and selection for desired traits, orby genetic engineering. Traits that might be optimized or alteredinclude, but are not limited to, traits involved in host rangedetermination, growth characteristics, improving phage production, orimproving traits important for the phage delivery processes. Thus, inanother aspect, the step of producing the infective phage panel includescreating engineered phages against the selected bacterial population.This will include phages created to have a broad host range. This may bethe product of directed genetic engineering, for example.

Collectively, the phages pooled together are referred to herein as theinfective phage multi-panel. Initial treatment of a target water systemwith the infective phage panel is ideally followed up by monitoring theeffects of treatment on the selected bacterial subpopulation. Overlonger periods of time, it may be necessary to alter the phage panel toconfront bacteria that have developed resistance mechanisms to theinfective phage panel. This is especially true if the phages isolatedabove ground and in the absence of salt are found to not be viable atdown-hole formation conditions. Additionally, new bacterial species maybegin to thrive in the absence of the initial selected bacterialsubpopulation. Thus, the need may arise to alter the infective phagepanel over time. New infective phage multi-panels may be created inresponse to either resistant strains or new bacterial populationscausing biofilm fouling or bio-corrosion. The effectiveness of theinfective phage panel is, in one embodiment, monitored by evaluatingchanges in phage and bacterial host populations within the system. Onecan either determine the presence of such bacterial populationsdirectly, or simply monitor the formation of new biofilms and thereoccurrence of bio-corrosion events.

Large Scale Phage Production

Phages are produced, in one embodiment, using a standard liquid lysatemethod. It should be noted that industrial scale phage production hasbeen achieved inadvertently by the dairy industry and historically bythe acetone/butanol fermentation industry, which demonstrates thefeasibility of aerobic and anaerobic phage production on this scale.

1. Prepare an exponentially (=OD600˜0.3) growing stock of the targethost bacteria in the volume of liquid corresponding to the desired finallysate volume. This is done by inoculating the media from a stationarystage liquid culture to a very low cell density (OD600˜0.01) andmonitoring growth spectrophotometrically until the desired OD isreached.2. Inoculate this culture with phage to a moi (multiplicity ofinfection=ratio of phage particles to individual host cells) of 0.1 to0.001.3. The culture is then incubated until lysis is observed; incubation istypically overnight but can take several days depending on the hostgrowth rate. At this point, the lysate is ready for purification of thephage particles away from both bacterial cell debris and the componentsof the culture media. This is accomplished first by vacuum filtrationthrough a filter series with the final pore size being 0.2 μm. Finally,tangential flow filtration will be used to replace components of themedia with 10 mM phosphate buffer and, if necessary, to concentrate thephage.

Since phages are notoriously hardy, they may be concentrated, freezedried and stored for long periods of time without loss of effectiveness.Phages may also be encapsulated with a coating that dissolves in water.This allows phage panels (cocktails) and multi-panels to be shipped toremote locations for use. It allows the manufacture to be made atoptimized central locations. While it is desirable that steps 1-6 beperformed “on location,” it is sometimes preferred that the manufactureof the large scale phage panel be centralized in locations where thenecessary equipment and resources are readily available.

In this specification, the invention has been described with referenceto specific embodiments. It will, however, be evident that variousmodifications and changes can be made thereto without departing from thebroader spirit and scope of the invention as set forth in the appendedclaims. The specification is, accordingly, to be regarded in anillustrative rather than a restrictive sense. Therefore, the scope ofthe invention should be limited only by the appended claims.

The invention claimed is:
 1. A process for remediation of souring causedby hydrogen sulfide in oil, gas and coalbed reservoirs comprising,providing a water pond or storage vessel or vessels, adding a solutionor solutions of bacteriophages virulent for target bacteria to water inthe provided pond or storage vessel and adding an effective amount ofthe water containing bacteriophage virulent for target bacteria from thepond or storage vessel into a reservoir wherein target bacteria arethose identified as existing in the water of the pond or storage vesselby the procedure comprising: a. Identifying target bacteria producinghydrogen sulfide from a sample or samples of the water to be used inhydrofracturing operation; b. Culturing target bacterial strains fromthe sample; c. Locating and isolating virulent phages for targetbacteria so identified; d. Preparing suitable quantity of identifiedphage multi-panel to treat the water in the pond or storage vessel. 2.The process of claim 1 wherein a solution of bacteriophage in solutionfrom the pond or storage vessel is injected into a reservoir and isalternated with injection of hydrofracturing water in the form ofsegmented injections into segments of the reservoir.
 3. The process ofclaim 1 wherein the bacteriophage are added to a pond or storage vesselby adding it slowly in discrete locations to allow localized areaselevated concentration of added bacteriophage and sufficient time foradded bacteriophage to lyse of target bacteria and thereby proliferatebacteriophage in said pond before large scale mixing.
 4. The process ofclaim 1 wherein the amount of solution or solutions added is that amountthat will result in a concentration of bacteriophage virulent for atleast one target bacteria in the water retained in an oil or gasreservoir after a hydrofracturing operation in which bacteriophage isalso injected with the hydrofracturing operation.
 5. The process ofclaim 1 wherein the effective amount of bacteriophage compriseindigenous bacteriophage isolated from bacteria contained in producedwater from the reservoir.
 6. The process of claim 1 wherein theconcentration and type of target bacteria in water recovered from thereservoir is monitored and virulent bacteriophages are prepared andadded to the pond or storage tanks to be effective against the type ofbacteria discovered from the monitoring.
 7. The process of claim 1wherein an effective amount of a first solution of bacteriophage isinjected into an oil or gas reservoir prior to injection of more highlypressurized hydrofracturing water in a hydrofracturing operation.
 8. Theprocess of claim 7 wherein the first solution will be about 0.01 to 10percent of the total hydrofracturing water injected.